Functional analysis of small-molecule and protein binding domains using monomeric streptavidin-based two-component biosensors
- 1Queensland University Of Technology
Abstract
Protein biosensors are important analytical and diagnostic tools that convert molecular recognition into measurable activity. Most common two-component biosensors require two non-competing recognition domains, yet binding affinity and specificity alone do not reliably predict their compatibility with the rest of the biosensor. We report a modular platform that exploits monomeric streptavidin (mSA2) as a surrogate recognition domain, enabling rapid functional evaluation of candidate binders in the context of biosensor scaffold without requiring a second ligand-specific binding protein. We established a chromatography-free protocol for recovering functional mSA2-based biosensor components from Escherichia coli. Using biotinylated small molecules methotrexate and vitamin D3, as well as heart failure protein biomarkers galectin-3 and S100A7 we demonstrate ligand-dependent reporter biosensor activation. The developed platform provides a scalable approach for rapid binder evaluation and biosensor prototyping, streamlining biosensor development using binding domains generated by directed evolution or computational protein design.
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BibTeX
@misc{mutschler2026functional,
title = {{Functional analysis of small-molecule and protein binding domains using monomeric streptavidin-based two-component biosensors}},
author = {R. Mutschler and Z. Guo and M. M. Fiorito and K. Alexandrov and Z. Cui},
howpublished = {bioRxiv},
year = {2026},
month = nov,
doi = {10.64898/2026.11.03.763809},
url = {https://www.biorxiv.org/content/10.64898/2026.11.03.763809v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Functional analysis of small-molecule and protein binding domains using monomeric streptavidin-based two-component biosensors
AU - R. Mutschler
AU - Z. Guo
AU - M. M. Fiorito
AU - K. Alexandrov
AU - Z. Cui
PY - 2026
DA - 2026/11/03
PB - bioRxiv
DO - 10.64898/2026.11.03.763809
UR - https://www.biorxiv.org/content/10.64898/2026.11.03.763809v1
AB - Protein biosensors are important analytical and diagnostic tools that convert molecular recognition into measurable activity. Most common two-component biosensors require two non-competing recognition domains, yet binding affinity and specificity alone do not reliably predict their compatibility with the rest of the biosensor. We report a modular platform that exploits monomeric streptavidin (mSA2) as a surrogate recognition domain, enabling rapid functional evaluation of candidate binders in the context of biosensor scaffold without requiring a second ligand-specific binding protein. We established a chromatography-free protocol for recovering functional mSA2-based biosensor components from Escherichia coli. Using biotinylated small molecules methotrexate and vitamin D3, as well as heart failure protein biomarkers galectin-3 and S100A7 we demonstrate ligand-dependent reporter biosensor activation. The developed platform provides a scalable approach for rapid binder evaluation and biosensor prototyping, streamlining biosensor development using binding domains generated by directed evolution or computational protein design.
ER -